PI: Irvine, Darrell J. Project Summary/Abstract: Strategies to promote the magnitude and quality of T cell and antibody responses following immunization have broad relevance for the development of new prophylactic and therapeutic vaccines for the treatment of cancer and infectious diseases. Recent studies, including work from our own laboratories, have demonstrated that the kinetic pattern of antigen and adjuvant exposure to lymphoid tissues has a substantial impact on the immune response to vaccination. However, active control over the temporal pattern of antigen/inflammatory cue delivery to lymph nodes is lacking in all current vaccine approaches. Here we propose an approach applying methods from synthetic biology to create nucleic acid-based vaccines where vaccine antigen/adjuvant expression dynamics can be controlled by (i) exogenous regulation by orally-available FDA-approved small molecule drugs or (ii) intrinsically programmed in genetic circuits carried by the RNA. Based on the promising features of RNA-based vaccines, in preliminary studies we established a lipid nanoparticle-delivered self- replicating alphavirus replicon RNA as the platform for these regulated vaccines. We will systematically study the impact of vaccine antigen and adjuvant kinetics on the immune response to vaccination, create pre- programmed vaccine kinetic patterns, and test the capacity of regulated replicons to enable single-shot vaccines with prime and boost controlled by an orally-available small molecule drug. Our specific aims are (1) To optimize small molecule-regulated expression of antigen and molecular adjuvants from RNA replicons, (2) To use the regulated replicon platform to define optimal kinetics of antigen and adjuvant expression during vaccination, (3) To design RNA-based replicon genetic circuits with pre-programmed temporal patterns, and (4) To determine factors limiting replicon expression lifetimes in vivo, and engineer strategies to prolong expression toward the goal of small molecule-regulated prime-boost regimens. These studies will lead to fundamental discoveries in basic immunology, provide a framework for rationally designing immunization regimens, and create technologies to practically implement them.